JMIR Publications

ABSTRACT

Background:

The development of new large-scale saliva pooling detection strategies can significantly enhance testing capacity and frequency for asymptomatic individuals, which could be crucial in containing SARS-CoV-2.

Objective:

This study aims to implement and scale up a SARS-CoV-2 screening method using pooled saliva samples for controlling the virus in critical areas while assessing its ability to detect asymptomatic infections.

Methods:

Between August 2020 and February 2022, our laboratory received a total of 928,357 samples. Participants collected a minimum of 1 mL of saliva using a self-sampling kit and registered samples in their smartphones. All samples were directly processed using the AutoMate™ 2550 for preanalytical steps and subsequently transferred to the Microlab® STAR and managed with HAMILTON Pooling software for pooling. The standard pool preset size was 20 samples but changed to 5 when the prevalence exceeded 2% in any group. The RT-PCR performed using the Allplex™ SARS-CoV-2 Assay until July 2021, followed by the Allplex™ SARS CoV-2 FluA/FluB/RSV for rest of study period.

Results:

Of 928,357 samples received, 887,926 samples (95.64%) were fully processed into 56,126 pools. 4,863 of these pools tested positive, detecting 5,720 asymptomatic infections. This This enabled a comprehensive analysis of pooling's impact on RT-PCR sensitivity and false negative rate (FNR). positive samples per pool (PPP) was collected. We take Ctref as the minimum Cycle threshold (Ct) of each Cts data set from one sample or pool and compared the Ctref results from pooled samples with those of the individual tests (ΔCtP). We then examining their distance to the expected offset due to dilution [ ΔΔCtP= ΔCtP- log2(Pool size)]. In this work, the ΔCtP and ΔΔCtP Global vs PPP1(one positive sample per pool) was 2.23 Vs 3.33 and -0.89 Vs 0.23. Hence, depending on the number of genes employed in the test and the size of the pool, we can evaluate FNR and effective sensitivity (1-FNR) of the configuration of the test. For our scenario (maximum 20 samples per pool, 3 target genes), statistical observation showed effective sensitivity above 99%. From an economic perspective, the focus lies in Pooling efficiency measured by individuals tested per test (PPT). The global PPT for this study was 8.66, reflecting savings exceeding 20 million euros based on our reagent prices.

Conclusions:

Our results demonstrate that, as expected, pooling reduces the sensitivity of RT-PCR, but with the proper pool size and multiple target genes, effective sensitivity can remain above 99%. Saliva pooling may be a useful tool for screening and surveillance in nonsymptomatic patients and help to control SARS-CoV-2 transmission. New studies are needed to evaluate the effectiveness of these strategies for SARS-CoV-2, as well as their application to other microorganisms or biomarkers detected by PCR. Clinical Trial: Not applicable