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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.
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• Assessment of the use of sample pooling in retrospective samples • A pool size of five samples had the highest sensitivity • Pooling prospective samples identified positive cases with cycle thresholds ranging from 16 7 to 39 4 Limited testing capacity has characterized the ongoing coronavirus disease 2019 (COVID-19) pandemic in Spain, hampering timely control of outbreaks and opportunities to reduce the escalation of community transmission This study investigated the potential to use sample pooling, followed by one-step retrotranscription and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to increase testing capacity for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Various pool sizes (five, 10 and 15 samples) were evaluated prior to RNA extraction followed by standard RT-qPCR for the diagnosis of COVID-19 The pool size achieving reproducible results with individual sample testing was subsequently used to assess nasopharyngeal samples in a tertiary hospital in August 2020 A pool size of five samples had higher sensitivity compared with pool sizes of 10 and 15 samples, showing a mean cycle threshold (Ct) shift of 3 5 [standard deviation (SD) 2 2] between the pooled test and positive samples in the pool Next, a pool size of five was used to test a total of 895 pools (4475 prospective samples) using two different RT-qPCR kits The Real Accurate Quadruplex corona-plus PCR Kit (PathoFinder) reported the lowest mean Ct shift [2 2 (SD 2 4)] between the pool and individual samples This strategy enables detection of individual positive samples in positive pools with Ct of 16 7–39 4 Grouping samples into pools of five for RT-qPCR resulted in an increase in SARS-CoV-2 testing capacity with minimal loss of sensitivity compared with testing each sample individually [ABSTRACT FROM AUTHOR] Copyright of International Journal of Infectious Diseases is the property of Elsevier B V and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder\'s express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )
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