A novel strategy for SARS-CoV-2 mass screening with quantitative antigen testing of saliva: a diagnostic accuracy study

Abstract

Background: Quantitative RT-PCR (RT-qPCR) of nasopharyngeal swab (NPS) samples for SARS-CoV-2 detection requires medical personnel and is time consuming, and thus is poorly suited to mass screening. In June, 2020, a chemiluminescent enzyme immunoassay (CLEIA; Lumipulse G SARS-CoV-2 Ag kit, Fujirebio, Tokyo, Japan) was developed that can detect SARS-CoV-2 nucleoproteins in NPS or saliva samples within 35 min. In this study, we assessed the utility of CLEIA in mass SARS-CoV-2 screening.

Methods: We did a diagnostic accuracy study to develop a mass-screening strategy for salivary detection of SARS-CoV-2 by CLEIA, enrolling hospitalised patients with clinically confirmed COVID-19, close contacts identified at community health centres, and asymptomatic international arrivals at two airports, all based in Japan. All test participants were enrolled consecutively. We assessed the diagnostic accuracy of CLEIA compared with RT-qPCR, estimated according to concordance (Kendall's coefficient of concordance, W), and sensitivity (probability of CLEIA positivity given RT-qPCR positivity) and specificity (probability of CLEIA negativity given RT-qPCR negativity) for different antigen concentration cutoffs (0·19 pg/mL, 0·67 pg/mL, and 4·00 pg/mL; with samples considered positive if the antigen concentration was equal to or more than the cutoff and negative if it was less than the cutoff). We also assessed a two-step testing strategy post hoc with CLEIA as an initial test, using separate antigen cutoff values for test negativity and positivity from the predefined cutoff values. The proportion of intermediate results requiring secondary RT-qPCR was then quantified assuming prevalence values of RT-qPCR positivity in the overall tested population of 10%, 30%, and 50%.

Findings: Self-collected saliva was obtained from 2056 participants between June 12 and Aug 6, 2020. Results of CLEIA and RT-qPCR were concordant in 2020 (98·2%) samples (Kendall's W=0·99). Test sensitivity was 85·4% (76 of 89 positive samples; 90% credible interval [CrI] 78·0-90·3) at the cutoff of 0·19 pg/mL; 76·4% (68 of 89; 68·2-82·8) at the cutoff of 0·67 pg/mL; and 52·8% (47 of 89; 44·1-61·3) at the cutoff of 4·0 pg/mL. Test specificity was 91·3% (1796 of 1967 negative samples; 90% CrI 90·2-92·3) at the cutoff of 0·19 pg/mL, 99·2% (1952 of 1967; 98·8-99·5) at the cutoff of 0·67 pg/mL, and 100·0% (1967 of 1967; 99·8-100·0) at the cutoff of 4·00 pg/mL. Using a two-step testing strategy with a CLEIA negativity cutoff of 0·19 pg/mL (to maximise sensitivity) and a CLEIA positivity cutoff of 4·00 pg/mL (to maximise specificity), the proportions of indeterminate results (ie, samples requiring secondary RT-qPCR) would be approximately 11% assuming a prevalence of RT-qPCR positivity of 10%, 16% assuming a prevalence of RT-qPCR positivity of 30%, and 21% assuming a prevalence of RT-qPCR positivity of 50%.

Interpretation: CLEIA testing of self-collected saliva is simple and provides results quickly, and is thus suitable for mass testing. To improve accuracy, we propose a two-step screening strategy with an initial CLEIA test followed by confirmatory RT-qPCR for intermediate concentrations, varying positive and negative thresholds depending on local prevalence. Implementation of this strategy has expedited sample processing at Japanese airports since July, 2020, and might apply to other large-scale mass screening initiatives.

Funding: Ministry of Health, Labour and Welfare, Japan.

Figure 1

Participant enrolment in the three study cohorts

Figure 2

Comparison of viral load between RT-qPCR and CLEIA in saliva samples (A) Histogram of CLEIA antigen concentration according to diagnostic outcome of RT-qPCR. Numbers of participants with each antigen concentration range are shown above each column. (B) Antigen concentration measured by CLEIA and Ct values from RT-qPCR were plotted according to symptomatic and asymptomatic status. W indicates Kendall's coefficient of concordance. Data were plotted for RT-qPCR-positive samples (n=89). A histogram of Ct values is also shown. RT-qPCR=quantitative RT-PCR. CLEIA=chemiluminescent enzyme immunoassay. Ct=cycle threshold. *Minimum antigen concentration was displayed as 0·01 pg/mL by the Lumipulse G1200 machine.

Figure 3

Diagnostic performance against antigen cutoff value Graphs were plotted by the cutoff values for antigen concentration. The solid line indicates point estimates and the dashed lines indicate 90% credible intervals. The cutoff value indicates the antigen concentration equal to and above which a sample is considered positive and below which a sample is considered negative. CLEIA=chemiluminescent enzyme immunoassay. RT-qPCR=quantitative RT-PCR. (CLEIA[+]|RT-qPCR[+])=CLEIA positivity given RT-qPCR positivity. (CLEIA[–]|RT-qPCR[–])=CLEIA negativity given RT-qPCR negativity.

Figure 4

Proportion of secondary RT-qPCR tests needed after initial CLEIA in a two-step strategy The proportion of secondary RT-qPCR tests needed (representing indeterminate results on CLEIA) against the lower cutoff value (ie, cutoff for CLEIA test negativity) was plotted for different probabilities of RT-qPCR positivity. The upper cutoff value (ie, for CLEIA test positivity) was set at 4·00 pg/mL. RT-qPCR=quantitative RT-PCR. CLEIA=chemiluminescent enzyme immunoassay.