key: cord-1021273-0smnxusm
authors: Alcoba-Florez, Julia; Gil-Campesino, Helena; García-Martínez de Artola, Diego; Díez-Gil, Oscar; Valenzuela-Fernández, Agustín; González-Montelongo, Rafaela; Ciuffreda, Laura; Flores, Carlos
title: Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling
date: 2020-11-19
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2020.11.155
sha: eead43c3455138c00ff86e4693eb058d26e540e4
doc_id: 1021273
cord_uid: 0smnxusm

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.

The SARS-CoV-2 pandemic causing COVID-19 continue imposing a heavy burden on healthcare systems worldwide because of a shortage of consumables and the demand for scaling up efficient screening approaches. To limit the escalation of cases and amplification of infections, increasing the capacities and developing alternatives to the one-step reverse transcription and real-time quantitative PCR (RT-qPCR) for regular testing of SARS-CoV-2 is key . We have assessed a direct heating method of nasopharyngeal (swab) samples to bypass the RNA extraction step for increasing the testing capacity (Alcoba-Florez et al. 2020a,b) .

In areas with low COVID-19 prevalence, such as the Canary Islands until June 2020 (Pollán et al. 2020) , testing of samples in pools is another approach that can efficiently increase SARS-CoV-2 testing capacity. Previous studies stated that pooling with a maximum of 10 samples would be desirable (Volpato et al. 2020) , with six being the optimal pool size for the target prevalence observed in the region (Regen et al. 2020) . Simulation studies support the value of testing on sample pools and the speed of reporting, while the impact on the sensitivity of tests is comparably smaller . Supporting this, a pooled test of SARS-CoV-2 of four samples has received Emergency Use Authorization from the USA Food and Drug Administration (FDA COVID-19 Update 2020).

Here we aimed to evaluate the RT-qPCR testing on pooled swab samples with the goal to demonstrate its feasibility as an option to increase SARS-CoV-2 testing capacity in the region. In a pilot stage, we used the Real Accurate Quadruplex corona-plus PCR Kit for testing 15 pools made by combining 5, 10, or 15 retrospective samples, each containing equal volumes of a SARS-CoV-2/COVID-19 positive sample and the respective amounts of negative samples to complete the pool size. In a validation stage, we used the two RT-qPCR kits available on swab samples from prospective subjects using the pooling size achieving optimal results. Then, individual samples from each positive pool were subjected to RNA extraction followed by RT-qPCR using the abovementioned methods to validate the results.

Differences between the pooled Ct and the positive sample Ct in the pool (Ct shift) for the two different RT-qPCR kits were assessed by Mann-Whitney U-test using J o u r n a l P r e -p r o o f the R v4.0.3 software. When more than a positive sample was present in the pool, the mean Ct of the positive samples was used in the calculation. Sensitivity and the 95% Confidence Interval (CI) was assessed with MedCalc (MedCalc Software Ltd.).

In a pilot study, detection of SARS-CoV-2 was achieved with pool size of five ( 

In this study, we describe the feasibility of RT-qPCR in pools of five swab samples with a minimal loss of sensitivity compared to the assessment of samples independently.

Consistent with these findings, using alternative RT-qPCR solutions and viral target J o u r n a l P r e -p r o o f probes others have recently demonstrated that pooling of up to 10 samples result in a slight shift in Ct (around 3), and therefore a drop of sensitivity (Das et al. 2020; Volpato et al. 2020) .

A major limitation of the study is that sensitivity was only evaluated in the pilot stage. However, despite the small Ct shift between the positive pools and the individual positive samples in the prospective study, a high impact of false negatives is not expected (Cherif et al. 2020) . Our data suggest that our sample pooling strategy is able to detect COVID-19 positive cases with Ct values 39.4. Therefore, our data support that RT-qPCR testing on pooled swab samples before RNA extraction is accurate, sensitive, and feasible as an option to efficiently increase SARS-CoV-2 testing capacity and for preparedness to test large amounts of people to control virus community transmission and sudden uncontrolled outbreaks. Pooling with compressive sampling designs or hypercube slicing algorithms that involve repetitive tests (Shental et al. 2020; Mutesa et al. 2020) , pooling of alternative less-invasive samples from the oral cavity (Watkins et al. 2020) , and the use of alternative testing approximations (Peto et al. 2020 ) may help to further lessen the impact of the dilution factor on pooling and continue increasing health capacity building.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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