Pooled Saliva Specimens for SARS-CoV-2 Testing

Abstract

We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69). The percentages of positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% confidence interval [CI], 65.8% to 90.5%) and 99.8% (95% CI, 98.7% to 100%), respectively. The percent positive agreement increased to 90.0% (95% CI, 74.4% to 96.5%) when considering only samples with moderate to high viral load (cycle threshold [C_T ] for the NP, ≤34). Pools of five saliva specimens were also evaluated on three platforms, bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche Cobas 6800. The average loss of signal upon pooling was 2 to 3 C_T values across the platforms. The sensitivities of detecting a positive specimen in a pool compared with testing individually were 94%, 90%, and 94% for the CDC 2019-nCoV real-time RT-PCR, Panther Fusion SARS-CoV-2 assay, and Cobas SARS-CoV-2 test, respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport medium for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Keywords: RT-PCR; SARS-CoV-2; asymptomatic screen; coronavirus; diagnostic test; nasopharyngeal swab; pooled saliva; saliva.

FIG 1

Comparison of cycle threshold (C_T) values of N1 for NP versus SAL specimens. (A) N1 C_T values for paired NP/MT and SAL samples (29 pairs). Pairs are connected by a line. The N1 C_T was set to 40 for samples for which N1 was not detected, indicating negative for SARS-CoV-2 RNA. The horizontal dashed line is at C_T = 40, the assay cutoff. P  < 0.001, calculated with the Wilcoxon matched-pair signed rank test. (B) A lower median viral load was seen for SAL specimens than the median C_T for NP/MT samples. The medians and interquartile ranges are 26 (21 to 34) for NP/MT and 31 (29 to 37) for SAL. P < 0.001. (C) RP C_T values for NP/MT and SAL specimens (424 pairs). The medians and interquartile ranges are 24 (23 to 25) for NP/MT and 22 (21 to 23) for saliva. The horizontal dashed line is at C_T = 40, the assay cutoff. P < 0.001, calculated with the Wilcoxon matched-pair signed rank test.

FIG 2

Comparison of cycle threshold (C_T) values for individual and pooled saliva specimens on different testing platforms. (A) C_T values for paired individual and pooled samples (easyMAG/ABI 7500) for 52 pairs. (B) C_T values for paired individual (easyMAG/ABI 7500) and pooled samples (Hologic Panther) for 41 pairs. (C) C_T values for paired individual (easyMAG/ABI 7500) and pooled samples (Roche COBAS 6800) for 50 pairs. For panels A to C, pairs are connected by a line. The horizontal dashed line is at C_T = 40, the assay cutoff. P < 0.001, calculated with the Wilcoxon matched-pair signed rank test. For panels A to C, the pooled C_T was set to 40 for samples for which N1 was not detected.