Accelerating the Laboratory Testing Capacity through Saliva Pooling Prior to Direct RT-qPCR for SARS-CoV-2 Detection

Abstract

The testing capacity of the laboratory is paramount for better control of the pandemic caused by SARS-CoV-2. The pooling method is promising to increase testing capacity, and the use of direct NAAT-based detection of SARS-CoV-2 on a non-invasive specimen such as saliva will ultimately accelerate the testing capacity. This study aims to validate the pooling-of-four method to quadruple the testing capacity using RNA-extraction-free saliva specimens. In addition, we intend to investigate the preferable stage of pooling, including pre- or post-heating. The compatibility of this approach was also tested on five commercial kits. Saliva specimens stored at −80 °C for several months were proven viable and were used for various tests in this study. Our findings revealed that pooling-of-four specimens had an overall agreement rate of 98.18% with their individual testing. Moreover, we proved that the pooling procedure could be conducted either pre- or post-heating, with no discordance and no significant difference in Ct values generated. When compared to other commercial detection kits, it demonstrated an overall agreement greater than 85%, which exhibits broad compatibility and ensures easy adaptability in clinical settings. This method has been proven reliable and increases the testing capacity up to fourfold.

Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting

“…If negative pooling is detected, all samples in the pool are assumed to be below the test’s detection limit. In contrast, if positive pooling is detected, each specimen is required to undergo further individual evaluation 11 .The pooled screening strategy could be implemented most effectively in low prevalence settings with a 5–6% positivity rate of infection 12 – 14 . Numerous studies have demonstrated that the pooling testing method results in high agreement on positive specimen detection and a comparable cycle threshold (Ct) value between pooled and individual specimens, although an expected slight loss of sensitivity has been shown, particularly when individual specimens have a low viral load 15 – 17 .…”

Development and validation of cost-effective SYBR Green-based RT-qPCR and its evaluation in a sample pooling strategy for detecting SARS-CoV-2 infection in the Indonesian setting

“…If negative pooling is detected, all samples in the pool are assumed to be below the test's detection limit. In contrast, if positive pooling is detected, each specimen is required to undergo further individual evaluation [12]. The pooled screening strategy could be implemented most effectively in low prevalence settings with a 5-6% positivity rate of infection [13][14][15].…”