Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling

Alcoba-Florez, J.; Gil-Campesino, H.; Garcia-Martinez de Artola, D.; Diez-Gil, O.; Valenzuela-Fernandez, A.; Gonzalez-Montelongo, R.; Ciuffreda, L.; Flores, C.

Objectives: Limited testing capacity has characterized the ongoing COVID-19 pandemic in Spain, hampering a timely control of outbreaks and the possibilities to reduce the escalation of community transmissions. Here we investigated the potential of using pooling of samples followed by one-step retrotranscription and quantitative PCR (RT-qPCR) to increase SARS-CoV-2 testing capacity. Methods: We first evaluated different sample pooling (1:5, 1:10 and 1:15) prior to RNA extractions followed by standard RT-qPCR for SARS-CoV-2/COVID-19 diagnosis. The pool size achieving reproducible results in independent tests was then used for assessing nasopharyngeal samples in a tertiary hospital during August 2020. Results: We found that pool size of five samples achieved the highest sensitivity compared to pool sizes of 10 and 15, showing a mean ({+/-} SD) Ct shift of 3.5 {+/-} 2.2 between the pooled test and positive samples in the pool. We then used a pool size of five to test a total of 895 pools (4,475 prospective samples) using two different RT-qPCR kits available at that time. The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct ({+/-} SD) shift (2.2 {+/-} 2.4) among the pool and the individual samples. The strategy allows detecting individual samples in the positive pools with Cts in the range of 16.7-39.4. Conclusions: We found that pools of five samples combined with RT-qPCR solutions helped to increase SARS-CoV-2 testing capacity with minimal loss of sensitivity compared to that resulting from testing the samples independently.